This invention relates to fluorometric systems for the detection of sample substances derived from biological fluid or tissue tagged with fluorochromes and to fluorometers adapted for more accurate measurements of surface mounted fluorescent samples. It is particularly useful in the detection of hormones, enzymes, drugs and other substances.
Most infectious diseases of bacterial or viral nature produce antibodies in the blood serum of the subject. This provides a degree of immunity against future assaults by the identical infectious agent or antigen. One method for detecting the presence of a particular antigen is to add to it a specific antibody which binds to the antigen. If the antibody has been previously tagged with a radioactive element (RIA technique) or a fluorescent dye, which does not interfere with its immunological properties, the coupled comples can be detected by an appropriate detector; and, in the case of the fluorescent additive, can be at best semiquantitatively measured, as is done in cases in the prior art on a microscope slide for visual inspection.
There are many reasons why RIA is not completely satisfactory. For example, in the presence of small quantities of antigen, only few counts per second can be detected. Since the "noise" of the system is proportional to the square root of the signal count, large errors in accuracy are made at low signal levels. Furthermore, radioisotopes have a limited shelf life due to half life decay, and require special licensing, handling and disposal.
Testing which relies on fluorescense tagging techniques, as heretofore known, has been qualitative, or at best, semiquantitative as an assay. In the area of the largest diagnostic use of fluorometry (i.e. immumofluorescense microscopy in which samples are typically mounted on a microscope stage and illuminated with an exciting wavelength) the fluorescence is observed through the stage with appropriate filters interposed to select the wavelength to be observed. Typically, the resultant observation is recorded by a laboratory technician as a comparative degree of fluorescence, for example, 0, +1, +2, +3, or +4 by comparison to known reference concentrations. In some instances, where blood titre or concentration of antibodies is the desired unknown, the technician prepares a number of slides; on each is a different concentration of the test material. Thus, the technician may estimate a +4 reaction in the microscope when the blood serum or the bacteria broth medium was diluted 1:4 in distilled water, or 1:16, or 1:128, etc. It would be of great advantage to medical and clinical authorities if a fluorometer could automatically and quantitatively read titre or concentration quickly and accurately, without the necessity of making serial dilutions.
To accomplish the foregoing known systems use a very powerful light source and cause a phenomenon known as photobleaching. Sometimes photobleaching is so rapid that the system has to be shut-off and allowed to return to chemical stability over a certain period of time before another attempt at a reading can be made. In addition, also, it is found that the light itself which is used is usually so powerful that it causes photochemical reactions and loss of signal. Attempts to mechanize and make systems of the foregoing type quantitative have primarily relied on the use of complicated electronic computing systems which observe the ratios of the variable being measured, compute the slope of fading or photobleaching effects that are observed and extrapolate these back to an assumed zero time.
Both liquid scanning and surface scanning fluorometers are known and will be discussed. Liquid scanning respect to fluorometers usually utilize a cuvette in which a liquid containing the substance is disposed, through which excitation light is passed and from which the emitted fluorescence is observed normally in a right angle configuration. It has been found that these systems are unsuitable for the present applications primarily because liquid systems and cuvettes themselves, apart from the sample being investigated all contribute very substantial background fluorescense, so that, unless a very high degree of careful chemical separation is utilized together with extremely well controlled materials selected to have low fluorescence in the wavelengths of interest, such systems are unsuitable. Attemps to adapt these instruments for surface measurements have not been particularily successful. Such systems are typically inefficient and have not provided for the descrete handling of individual samples. In general past fluorometers and RIA systems have been unduly sensitive to background and non-sample oriented signals. There is therefore, a need for a new and improved fluorometric system.